Project Summary Immunoregulation, indicated by a balanced expansion of effector T-cell populations and regulatory T cells (Treg), is known to be driven by microbial signals, mainly by organisms with which mammals coevolved, including: (i) the commensal microbiota, which have been altered by the Western lifestyle, including a diet that is commonly low in microbiota-accessible carbohydrates; (ii), pathogens associated with the ?old infections? that were present throughout life in evolving human hunter-gatherer populations; and (iii) organisms from the natural environment with which humans were inevitably in daily contact (and so had to be tolerated by the immune system). Immunoregulation is thought to be compromised in modern high-income settings due to reduced contact with these three categories of organisms. A failure of immunoregulation, attributable to reduced exposure to the microbial environment within which the mammalian immune system evolved, is thought to be one factor contributing to recent increases in stress-related and chronic inflammatory disorders in high-income countries. Immunization with one of these ?old friends?, in the form of a heat-killed preparation of Mycobacterium vaccae, a nonpathogenic, environmental saprophyte with anti-inflammatory and immunoregulatory properties, has been shown to increase stress resilience in mice, as measured by prevention of stress-induced increases in anxiety, prevention of stress-induced exaggeration of spontaneous colitis and prevention of chemically induced colitis in a model of inflammatory bowel disease. However, the mechanisms through which M. vaccae mediates is stress protective effects on behavior, at the level of the central nervous system, are not known. Our preliminary data demonstrate that immunization with M. vaccae increases expression of the anti-inflammatory cytokine, interleukin (IL)-4, in the rat brain, prevents stress- induced priming of hippocampal microglia, and prevents stress-induced decreases in juvenile social investigation in rats. In this R21 proposal, we propose to determine, using male and female rats, if the stress- protective effects of immunization with M. vaccae, as measured by prevention of inescapable stress (IS)- induced increases in anxiety, conditioned fear, and escape deficits, and prevention of priming of hippocampal microglia, are dependent on increases in IL-4. We will also determine, for the first time, if IL-4 is sufficient to protect against IS-induced behavioral deficits. Finally, we will determine if the stress-protective effects of immunization with M. vaccae involve inhibition of specific neural circuits involved in mediating the behavioral sequelae of IS, including locus coeruleus, lateral habenula, and bed nucleus of the stria terminalis projections to the serotonergic dorsal raphe nucleus. Together, the proposed studies will provide the first detailed investigation of neural mechanisms through which immunization with M. vaccae promotes stress resilience.